Orofacial clefts are common congenital malformations which require long-term treatment and patient care. They are usually classified as either cleft lip with or without cleft palate (CL/P) or cleft palate only (CP), based on differences in embryological development [1, 2]. Approximately 70% of cases of orofacial clefting occur as isolated entities with no other apparent cognitive or craniofacial structural abnormalities and are commonly termed ‘isolated, non-syndromic CL/P (NSCL/P)’; while many syndromic forms have simple Mendelian patterns of inheritance and therefore are amenable to disease gene identification [3–5]. Previous genetic and epidemiological studies have indicated that the causes of nonsyndromic CL/P are multifactorial, with both genetic and environmental factors contributing to the phenotype [6–9].

Of the large number of candidate genes thought to contribute to orofacial clefting, Interferon Regulatory Factor 6 (IRF6) is the only gene that has shown a convincing degree of consistency across studies [10, 11]. Mutations in IRF6 were first identified in the autosomal dominant van der Woude syndrome (VWS; OMIM 119300), which is traditionally recognized as a monogenic syndrome characterized by lip pits, clefting of the primary or secondary palate, and hypodontia [12]. Van der Woude syndrome (VWS) is estimated as most common syndromic form of oral clefts, accounting for 2% of all cases of cleft lip and palate, and has the most closely similarities in phenotype with that of common non-syndromic forms [13–15]. Up to date, at least 200 different mutations in the IRF6 gene have been described with the majority being protein truncation mutations (nonsense and frameshifts) or missense mutations [16]. Given the overlapping phenotype of VWS with isolated CL/P, in searching the gene for mutations, non-pathogenic variants in IRF6 gene have also been found to be significantly associated with nonsyndromic oral clefting in many different populations and ethnic groups [10, 17–23].

In order to determine the contribution of mutations in IRF6 to oral clefts in the Taiwanese population, we applied a combination of targeted, PCR-based experimental approaches (including polymerase chain reaction (PCR), Denature High Performance Liquid Chromatography (DHPLC), Multiplex ligation-dependent probe amplification (MLPA), TOPO cloning and DNA sequence analysis) for a cohort of oral cleft patients from Craniofacial center of Chang Gung Memorial Hospital.

We have identified 11 different IRF6 mutations in VWS patients and none in 44 of the non-syndromic multiplex families and 80 non-syndromic oral cleft patients. In the present study, all affected members were heterozygous for their respective mutation and five of these mutations (p.Tyr97Cys, p.Gln120HisfsX24, p.Glu136fsX3, p.Thr291Pro, and p.Trp323X) have not been reported in the literature previously.

Interferon Regulatory Factor 6 (IRF6) is a member of transcription factors that contain a highly conserved helix-turn-helix DNA-binding domain and a less conserved SMAD-IRF-binding domain [12]. It has been suggested to be important contributor to orofacial development since mutations of the IRF6 gene have been found in Van der Woude (VWS) and popliteal pterygium syndromes (PPS), two disorders that can clinically resemble an isolated cleft lip and palate [12]. They were detected in approximately 70% of VWS and 97% of PPS cases [16]. Microdeletion of 1q32–q41 is relatively rare, and only a few cases have been reported in the medical literature [24–26]. While the majority of the VWS mutations were spread over exons 3, 4, 7, 8 and 9, the majority of PPS mutations were concentrated in exons 3 and 4 [16, 27]. Basically, our results support a non-random distribution of IRF6 mutations in VWS. More recently, patients with nonsyndromic oral clefts have also been reported to have IRF6 mutation [27–30]. Two authors have observed IRF6 mutations among families with mixed cleft phenotype, but they were unable to exclude VWS completely [28, 29]. Therefore, molecular differentiation between these overlapping cleft syndromes is absolutely essential before counseling can be given to family members.

The incidence of VWS among cleft patients in our population is 0.73 to 0.98 per 100,000, which is lower than those reported from other studies [31]. The result of IRF6 mutation screening for our VWS is also low (57.89%). All these VWS cases were selected from cleft patients, thus VWS patients presenting only with lower lip pits were not included. The patients in our series had more severe types of cleft, with a higher incidence of bilateral complete cleft lip and palate than given in other reports. The size, shape, and depth of the pits varied among patients beyond oral cleft phenotypes. The associated anomalies found in our series included hemangioma, hypertelorism, syngnathism, protruding ears, ventricular septal defect, toe and foot anomaly, and undescended testis. All the differences in the distribution of the different types of cleft, the penetrance, and familial occurrence between our study and other reports may be explained by ethnic and/or nongenetic environmental factors.

Lip and palate formations are the consequence of a complex processes that involves cell proliferation, cell differentiation, cell adhesion, and apoptosis. In theory, failure anywhere in these processes can lead to clefts [2, 32]. We hypothesize that IRF6 may play different roles in combination with other genetic and/or environmental factors during the embryonic development [33, 34]. Information regarding the profile of gene-gene or gene-environment interaction or network for the craniofacial development should also be considered. Meta analysis of mutation-proved IRF6-related phenotypes might provide another useful indicator for the diagnosis of orofacial cleft in the near future.

This is one of the largest single reports of IRF6 mutation screens applying a combined targeted mutation analysis. Our results confirm the crucial role of IRF6 in the VWS patients and further work is needed to explore for its function in the non-syndromic oral cleft with vary clinical features.